During the past decades lactobacilli have widely been used as probiotics in functional foods. Food can be regarded as functional, if beyond adequate nutritional components it contains some natural additives (pre- or probiotics), which beneficially affect one or more target functions in the body, either improving the state of health and well-being and/or reducing disease risk. Probiotics are live microorganisms, which, when administered in adequate scientifically proven amounts, confer a health benefit on the host. Probiotic products may be conventional foods or dietary supplements. Currently a probiotic product is a strain-specific preparation targeting several host functions (anti-infectious, morphologic, immunologic, metabolic) in order to improve health by either supporting host physiologic activity or by reducing the risk of disease.
Probiotics are often used for enhancement of organisms' defence capability.
Enhancement of organisms' natural immunity has become essential in connection with the ageing of population and diseases connected with immunodeficiency (HIV infection, tissue transplantation induced immunosuppression). The cause of all of mentioned diseases is the decrease of the capability of several physiological functions of the organism (Timiras, P. S. Physiology of aging: standards for age-related functional competence In: Comprehensive Human Physiology. Greger, R (edt)/Windhorst, U (eds) Springer Verlag, 1996; pp 2391-2405).
The permeability of the intestinal mucosa frequently increases on the background of chronic inflammation. Microbial metabolites play essential role in the integrity of mucosa, e.g. short chain fatty acids (SCFA), produced by lactic acid bacteria in the colon in the case of fiber (substances of plant origin) rich diet (Roy C C, Kien C L, Bouthillier L., Levy E. Short chain fatty acids: ready for prime time?Nutr. Clin. Pract., 2006; 21:351-366).
For enhancement of mucosal barrier besides short chain fatty acids also polyamines are essential. Polyamines are linear aliphatic compound, in which amino acids are situated along the structure. Putrescine, spermidine and spermine belong to polyamines (Larqué, M., Sabater-Molina, S. Zamora E. Biological significance of dietary polyamines. Nutrition 2007; 23(1): 87-95). Polyamines are produced by decarboxylation from amino acids ornithine and arginine. Putrescine is produced straight from omithine; arginine, is primarily converted into agmantine which is then converted into putrescine (Halaris A, Plietz, Agmatine: metabolic pathway and spectrum of activity in brain. CNS Drugs, 2007; 21: 885-90).
The physiological impact of polyamines is targeted to cell growth and differentiation, regulation of immune cells and inflammatory response, and several other effects. Polyamines possess the ability to induce apoptosis, avoiding the hyperproliferation of epithelium and destruction of primary cancer cells (Moinard C, Cynober L, De Bandt J P Polyamines: metabolism and implications in human disease. Clin Nutr. 2005; 24: 184-197). Polyamines are produced endogenously or they are obtained actively from food.
In the case of the damage of epithelial cells, the production of polyamines by the intestinal microflora is considered one of the compensatory mechanisms for modification of immune response and apoptosis regulation. Lactobacilli comprise majority of microflora of the proximal colon. Lactobacilli produce polyamines through decarboxylation of amino acids, particularly at the high pH of the intestinal content (Lonvaud-Funel A, Biogenic amines in wine: role of lactic acid bacteria. FEMS Microbiol. Letters, 2001: 199: 9-13). On the other hand, strains of Lactobacillus acidophilus utilize putrescine and reduce odour of faces (WO 2008/019887, BASF AG).
Probi AB Estonian patent EE03597 discloses pharmaceutical composition that contains L. plantarum strains 299 and 299v together with arginine for prevention translocation of intestinal microbes during liver injury. Also in this patent no information is available concerning the end products of arginine utilization (putrescine, cadaverine, tyramine, enhancement of NO or antioxidativity) by L. plantarum, which are responsible for aforementioned effect. In close relationship with previous patent another Probi AB patent EE04097 does not disclose the polyamines or NO production ability of mentioned L. plantarum strains.
It has been demonstrated, that Lactobacillus rhamnosus GG could enhance NO production in the epithelial cells of the intestine or by proinflammatory cytokines and it has been indicated, that beneficial effects of Lactobacillus rhamnosus GG could be due to the production of NO by macrophages and epithelial cells (Korhonen K, Reijonen T M, Remes K, Malmstrom K, Klaukka T, Korppi M. Reasons for and costs of hospitalization for paediatric asthma: A prospective 1-year follow-up in a population-based setting. Pediatr Allergy Immunol 2001: 12:331-338). It has been demonstrated, that NO protects mucosa for damages and excessive permeability, arising after reperfusion (Payne D, Kubes P. Nitric oxide donors reduce the rise in reperfusion-induced intestinal mucosal permeability. Am J Physiol. 1993: 265 (1 Pt 1):G189-G195).
USA patent application US20060078595 (Friesland Brands B.V.) discloses method to avoid the excessive permeability of the intestinal barrier in newborns by glutamate and its precursors as well as by polyamines spermidine, spermine, putrescine in the case of different syndromes (malnutrition, allergy, sepsis, translocation of microbes, endotoxemia, viral diarrhoea). Lacobacillus Reuteri (BIOGAIA) served as glutamate source.
Polyamine spermidine has inflammation-lowering property. It has been demonstrated that spermidine, when added to human monocytes stimulated with lipopolysaccharides, inhibits effectively the synthesis of TNF, IL-1, IL-6 and other proinflammatory cytokines (Zang M, Caragine T. Wong H et al. Spermine inhibits proinflammatory cytokine synthesis in human mononuclear cells: a counter regulatory mechanism, that restrains the immune response. J. Exp. Med., 1997, 185: 1759-1768). Matsumoto with co-authors described the suppression of proinflammatory cytokine synthesis (Matsumoto M, Ohisshi H, Benno Y Impact of LKM512 yoghurt on improvement of intestinal environment of the elderly. FEMS immunol. Medical Microbiol, 2001; 31:181-186). Bifidobacterium lactis LKM512 comprising yoghurt administration to elderly decreased the glukoprotein haptoglobuline caused inflammatory acute phase response due to IL-1, Il-6 and TNF-alfa, but the probiotic administration was also accompanied by decrease of mutagenicity of the intestinal epithelial cells. At the same time it is evident, that different lactic acid bacteria incl. lactobacilli species and strains differ by their ability to induce pro- and anti-inflammatory cytokines and non-specific cellular immune response. Up to now, no lactobacillus species/strain has been described, which would be able to produce physiologically relevant amounts of polyamines, which could be detected in urine after the consumption of this particular strain comprising composition and which promote simultaneously the adaptive activation of immunocytes due to interleukin IL-6.
Proinflammatory cytokine IL-6 synthesis has been described after 24 h of stimulation with different strains of Bifidobacterium animalis and Lactobacillus rhamnosus (Miettinen M., Vuopio-Varkila J, Varkila K. Synthesis of human tumour necrosis factor alpha, interleukin-6 and interleukin-10 is induced by lactic acid bacteria. Infection and Immunity, 1996, 64:5403-5408). It is important to observe inflammation markers like counts of leucocytes (WBC) and amount of CRP in sera on the induction of IL-6 (Kiecolt-Glaser J K, Preacher K J, MacCallum R C et al. Chronic stress and age-related increases in the proinflammatory cytokine IL-6, PNAS, 2003; 100:9090-9095) to avoid the overproduction of IL-6. Aforementioned is associated with cardiovascular diseases, arthritis, type II diabetes, cancer, periodontal diseases, cachexy and decrease of organisms functions (Rose-John S., J. Scheller, G. Elson, and S. A. Jones. Interleukin-6 biology is coordinated by membrane-bound and soluble receptors: role in inflammation and cancer J. Leukoc. Biol., Aug. 1, 2006; 80 (2): 227-236).
Lactobacillus plantarum is a widely spread representative of the genus Lactobacillus. Aforementioned lactobacillus species is present on fermented plants (sauerkraut, pickles, and silage), fermented dairy/meat products (cheese, salami) as well as in human gastrointestinal tract. Lactobacillus plantarum is able to reorganize its metabolism according to environmental conditions.
Probiotic Lactobacillus plantarum is available in probiotic foods as well as in food supplements (e.g. Lactobacillus plantarum 299v DSM 9843, Probi AB, Sweden, Skånemejeriers' ProViva probiotic brand in Sweden or as one of the components in bacterial composition VSL#3 (VSL Pharmaceuticals, Inc. USA). WO2007/108764 (Probac AB) discloses the action mechanisms of Lactobacillus plantarum strains, which are able to enhance immunotolerance in the case on autoimmune coeliac disease.
Cheese as a probiotic carrier has several controversial aspects. Incorporation lactobacilli of human origin into a food product different from other milk-based products and having a long ripening period could be complicated. At the same time fat and protein-rich cheese matrix protects a probiotic microbial strain throughout the passage of gastrointestinal tract better than other milk products (yoghurt, kefir). Antimicrobial and antioxidative probiotic cheese has been produced by using Lactobacillus fermentum ME-3 (DSM 14241) (Estonian patent EE04580, Russian patent RU2284354, U.S. Pat. No. 6,190,879). European patent EP1064857B1 (Snow Brand Milk Products Co Ltd., 2004) discloses methods for production substances incl. putrescine with lactobacilli, bifidobacteria and propionibacteria from Gouda cheese milk ultrafiltration. These methods are either polymerization reactions for incorporating putrescine into casein or vice versa—purification of these compounds by ultrafiltration, that have already been produced into milk, methods are different from this one described in present invention, where putrescine, that has been produced by lactobacilli into milk is still present in cheese after 30 days of ripening. Various non-starter lactobacilli have been described (Lactobacillus paracasei, Lactobacillus curvatus), which are able to gain energy for proliferation from ornithine (ornithine is released from milk casein arginine) after depletion of carbohydrates (Laht T.-M., Kask S., Elias P., Adamberg K., Paalme T. Role of arginine in the development of secondary microflora in Swiss-type cheese. Int. Dairy Journal, 2002, 12: 831-840).
Till now no lactobacillus species/strain have been described, the culture of which produces NO and additionally physiologically relevant amounts of polyamines in food product, whereas the latter could be detectable in urine after the consumption of this strain comprising food product (cheese) or composition and that is able to regulate through polyamines the apoptosis of intestinal epithelium and increase the count of the mucosal lymphfollicles and blood monocytes, regulating the condition of mucosa by NO and antioxidative compounds and to enhance the activation of immune cells particularly the activation of macrophages by central interleukine.
One of the factors leading to cardiovascular disease (CVD) is abnormally elevated cholesterol level. Recently, the view of high cholesterol as damaging agent has been reverted to abnormality of its particles, particularly low density lipoprotein-cholesterol (LDL-c). LDL-c accounts 60-70% of total cholesterol. LDL-c particles carry cholesterol, triglycerides, fat-soluble vitamins and antioxidants. The LDL-cholesterol is an important modulator for prevention of atherosclerosis and maintenance of cardiovascular health. Thus, the LDL-c is widely recognized as an established cardiovascular risk marker. The close relation between mucosal epithelial cells of host gut and microbiota is of utmost importance for health. Among indigenous microbiota of gastrointestinal tract (GIT) the lactic acid bacteria assimilate cholesterol from dietary products (Gilliland, S. E., Nelson, C. R., Maxwell, C., Assimilation of cholesterol by Lactobacillus acidophilus. Appl Environ Microbiol 1985; 49, 377-381). In patent of Cuñé Castellana, 2009 (EP2485743131; AB Probiotics S.A.) Lactobacillus plantarum strains CECT 7528, CECT 7526 and CECT 7529 as single or in composition demonstrated both in vitro and in vivo the cholesterol lowering ability. These strains have bile salt hydrolases (BHS) activity, also the antagonistic activity to inhibit the growth of pathogenic strains (Salmonella enterica Enteritidis, Salmonella enterica Typhimurium, Yersinia pseudotuberculosis, Clostridium perfringens, Clostridium ramnosus, Enterococcus faecalis) and can be used as probiotic bacteria.
Pereira et al. (Pereira, I). I., McCartney, A. L., Gibson, G. R., An in vitro study of the probiotic potential of a bile-salt-hydrolyzing Lactobacillus fermentum strain, and determination of its cholesterol-lowering properties. Appl Environ Microbiol. 2003; 69, 4743-4752) have demonstrated the role of short-chain fatty acid concentrations, specifically the molar proportion of propionate and/or bile salt deconjugation as the major mechanism involved in the purported cholesterol-lowering properties of L. fermentum. 
However, the effect of Lactobacillus spp strains on levels of serum cholesterol (and cholesterol fractions) is strain-specific and dependant on the origin and properties of a certain strain (Tanaka, H., K. Doesburg, T. Iwasaki and I. Mireau, Screening of lactic acid bacteria for bile salt hydrolase activity. J. Dairy Sci. 1999; 82: 2530-2535).
Disturbed Microbial Ecology of Gut
The colonic microbiota is well stabilised and due to mucosal microbiota it does not change easily. But it is well known that the application of broad-spectrum antimicrobial preparations for treatment of infections and inflammatory complications may cause profound imbalance among GI microbiota.
Clostridium difficile Infection
Clostridium difficile was identified in the 1970's as the causative agent of antibiotic associated diarrhoeae. The anaerobic spore-forming intestinal pathogen Clostridium difficile is spread in hospitals and elderly homes (Britton, R. A., Young, V. B. Interaction between the intestinal microbiota and host in Clostridium difficile colonization resistance. Trends Microbiol. 2012; 20, 313-319). C. difficile infection is initiated by infection with C. difficile spores. Endospore production is vital for the spread of Clostridium difficile infection. In order to cause disease, these spores must germinate and return to vegetative cell growth (Burns D. A, Heap J. T. Minton N. P. Clostridium difficile spore germination: an update. Res Microbiol. 2010; 161(9):730-4). C. difficile elicits disease through the actions of secreted toxins, which are produced by vegetative cells, not by spores.
In a quarter of patients (25%) infected with C. difficile develop serious sequela such as pseudomembraneous colitis (PMC). C. difficile-associated diarrhea (CDAD) increases mortality rates, lengthens hospitalization and dramatically increases overall health care costs. Clostridium difficile infection recurs in about 20% of patients, and increases to 40% and 60% with subsequent recurrences (Kelly, C. P., LaMont, J. T. Clostridium difficile—more difficult than ever. N Engl J Med. 2008; 359, 1932-1940). Antimicrobial treatment disrupts the complex balance of diverse microorganisms and is a key factor in the pathogenesis of C. difficile colonization and disease. Preservation and restoration of the microbial diversity could represent novel strategies. The crucial moment in prevention and treatment of this disease is to find the possibility to reconstitute the alteration of intestinal microbiota during and after antibiotic therapy with various regimens incl. administration of probiotics. Most probiotics colonize the gut temporarily, produce bactericidal acids and peptides and promote “competition” among microbes by competing for nutrients and epithelial adhesion. These effects appear to reduce the favourability of the environment for C. difficile. Previous studies suggest that probiotics for prevention of CDI include combination L. acidophilus and L. casei, S. boulardii, or L. rhamnosus. In addition, a dosage of >109 cfu/day is more effective than lower doses.
The antimicrobial activity of probiotic strains is one of the suggested mechanisms for competition with C. difficile. Lactic acid bacteria produce short chain fatty acids that lower the pH of the local gut environment as well as prevent the adhesion of C. difficile (McFarland, L. V., Beneda, H. W., Clarridge, J. E., Raugi, G. J. Implications of the changing face of C. difficile disease for health care practitioners. Am J Infect Control. 2007; 35, 237-253). Next, the possibility for intestinal barrier protection with probiotics may result in interfering with the binding of C. difficile toxins A and B to colonic epithelial cells thus stabilizing gut permeability and inhibiting development of pseudomembranes on epithelia of gut.
Strain-specificity of lactobacilli is an important factor to take into consideration when looking for potential probiotics in the prevention of C. difficile infection or binding the C. difficile toxins (Tejero-Sariñena S., Barlow J., Costabile A, Gibson G. R., I. Rowland Antipathogenic activity of probiotics against Salmonella Typhimurium and Clostridium difficile in anaerobic batch culture systems: Is it due to synergies in probiotic mixtures or the specificity of single strains?Anaerobe 2013: 24; 60-65).
Furthermore, some clinical trials could not reach statistical evidence to demonstrate the effect for the prevention of Clostridium difficile associated diarrhea (CDAD) of certain probiotics. The authors of a large, randomized trial including 2941 elderly adults with antibiotic exposure noted that those who received probiotics (a multistrain preparation of Lactobacillus acidophilus and Bifidobacterium bifidum) did not show a risk reduction for CDI (RR 0.71; 95% CI 0.34-1.47; p=0.35) (Allen, S. J., Wareham, K., Wang, D., Bradley, C., Hutchings, H., Harris, W., Dhar, A., Brown, H., Foden, A., Gravenor, M. B., Mack, D. Lactobacilli and bifidobacteria in the prevention of antibiotic-associated diarrhoea and Clostridium difficile diarrhoea in older inpatients (PLACIDE): a randomised, double-blind, placebo-controlled, multicentre trial. Lancet, 2013: 382, 1249-1257). After years of trials with different probiotics for treatment of CDI the strain, dose, and duration of probiotics are still under discussion (Naaber, P., Mikelsaar, M., Interactions between Lactobacilli and antibiotic-associated diarrhea. Adv Appl Microbiol, 2004: 54, 231-260).
Xylitol Application
Xylitol is a 5-C sugar alcohol, e.g. pentitol, and is found in plants, fungi and algae. Xylitol is an important intermediate product in mammalian carbohydrate metabolism; i.e. human blood contains up to 8×10−5 M of xylitol. Consumed xylitol is not absorbed completely and the unabsorbed part can be used as a dietary fibre for bacterial fermentation to convert xylitol to short fatty acid chains utilized in energy pathways. Xylitol influences the growth of some species of gut microbiota in the large intestines stimulating the growth and activities of indigenous microbiota. One gram of xylitol contains 2.4 kcal as compared to one gram of glucose which has 3.87 kcal. Xylitol is advertised as “safe” for diabetics and individuals with hyperglycaemia (Talbot J. M., K. P. Fisher The Need for Special Foods and Sugar Substitutes by Individuals with Diabetes Mellitus. Diabetes Care 1978: 1; 231-240).
In our previous studies on Caco-2 cell lines we have discovered that 1% xylitol prevented the adhesion of vegetative cells of C. difficile reference strain VPI 10463, seemingly blocking the receptors on cells. In an applied hamster model (Naaber, P., Lehto, E., Salminen, S., Mikelsaar, M. Inhibition of adhesion of Clostridium difficile to Caco-2 cells. FEMS Immunol Med Microbiol. 1996: 14, 205-209) 1 ml of 20% xylitol solution together with Lactobacillus rhamnosus GG significantly protected animals from development of severe enterocolitis (Naaber, P., Lehto, E., Salminen, S., Mikelsaar, M., 1996. Inhibition of adhesion of Clostridium difficile to Caco-2 cells. FEMS Immunol Med Microbiol, 14, 205-209). In these experimental studies with xylitol in combination with probiotic L. rhamnosus GG the vegetative cells of Clostridium difficile, precultivated in laboratory anaerobic environment, have applied for inoculation of cell cultures or hamsters.
In opposite, in clinical practice or elderly home the infection develops from inoculation with C. difficile extremely resistant spores surviving in the aerobic environment of these facilities. The spores start to germinate inside the intestine of host.
Some authors have postulated that in the animal model some of sugars similarly to glucose could block the expression of toxins A and B of C. difficile (Karlsson, S., Burman, L. G., Akerlund, T. Induction of toxins in Clostridium difficile is associated with dramatic changes of its metabolism. Microbiology, 2008: 154, 3430-3436).
There is still a need for probiotic strains effective in enhancing of cellular immunity, decreasing LDL-cholesterol as well as in lowering the risk of Clostridium difficile infection.